scholarly journals Evaluating the Effects of Disinfectants on Bacterial Biofilms Using a Microfluidics Flow Cell and Time-Lapse Fluorescence Microscopy

2020 ◽  
Vol 8 (11) ◽  
pp. 1837
Author(s):  
Milos Legner ◽  
James Jonkman ◽  
Dean Swift
Keyword(s):  
Fluorescence Microscopy ◽  
Bacterial Species ◽  
Bacterial Biomass ◽  
Time Lapse ◽  
Flow Cell ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Microscopy Imaging ◽  
Microscopy Method ◽  
Time Lapse Imaging

A commercially available microfluidics flow cell was utilized together with widefield fluorescence microscopy to evaluate the effects of disinfectants on bacterial strains. The flow cell’s inner surface supports the formation of biofilms of numerous bacterial species. The modular setup of the flow cell accessories allows connection to syringes, pumps and collection vials, facilitating aseptic experiments in a controlled fluidics environment which can be documented with precisely timed microscopy imaging. The flow cell is inoculated with a suspension of bacteria in a nutrient medium and incubated for several days allowing bacterial cells to form a biofilm. Shortly before performing an assay, the biofilm is labelled with a dual-fluorescent DNA probe which distinguishes unharmed and damaged bacteria. Then a disinfectant sample (or control) is gently injected and time-lapse imaging is used for quantifying the course of bacterial biomass response. We use a simplified widefield microscopy method that allows intensive recording and quantification of time series of two-dimensional frames for tracking the course of disinfectant action on a variety of microbial strains. This procedure has potential for the rapid evaluation of novel products.

scholarly journals Weathering-Associated Bacteria from the Damma Glacier Forefield: Physiological Capabilities and Impact on Granite Dissolution

2010 ◽  
Vol 76 (14) ◽  
pp. 4788-4796 ◽  
Author(s):  
Beat Frey ◽  
Stefan R. Rieder ◽  
Ivano Brunner ◽  
Michael Plötze ◽  
Stefan Koetzsch ◽  
...  
Keyword(s):  
Oxalic Acid ◽  
Bacterial Species ◽  
Soil Formation ◽  
Mineral Dissolution ◽  
Microbial Colonization ◽  
Swiss Alps ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Mountain Areas ◽  
Glacier Forefield

ABSTRACT Several bacterial strains isolated from granitic rock material in front of the Damma glacier (Central Swiss Alps) were shown (i) to grow in the presence of granite powder and a glucose-NH4Cl minimal medium without additional macro- or micronutrients and (ii) to produce weathering-associated agents. In particular, four bacterial isolates (one isolate each of Arthrobacter sp., Janthinobacterium sp., Leifsonia sp., and Polaromonas sp.) were weathering associated. In comparison to what was observed in abiotic experiments, the presence of these strains caused a significant increase of granite dissolution (as measured by the release of Fe, Ca, K, Mg, and Mn). These most promising weathering-associated bacterial species exhibited four main features rendering them more efficient in mineral dissolution than the other investigated isolates: (i) a major part of their bacterial cells was attached to the granite surfaces and not suspended in solution, (ii) they secreted the largest amounts of oxalic acid, (iii) they lowered the pH of the solution, and (iv) they formed significant amounts of HCN. As far as we know, this is the first report showing that the combined action of oxalic acid and HCN appears to be associated with enhanced elemental release from granite, in particular of Fe. This suggests that extensive microbial colonization of the granite surfaces could play a crucial role in the initial soil formation in previously glaciated mountain areas.

scholarly journals Critical Assessment of Methods to Quantify Biofilm Growth and Evaluate Antibiofilm Activity of Host Defence Peptides

Biomolecules ◽  
2018 ◽  
Vol 8 (2) ◽  
pp. 29 ◽  
Author(s):  
Evan Haney ◽  
Michael Trimble ◽  
John Cheng ◽  
Quentin Vallé ◽  
Robert Hancock
Keyword(s):  
Bacterial Species ◽  
Growth Conditions ◽  
Host Defence ◽  
Detection Methods ◽  
Antibiofilm Activity ◽  
Bacterial Cells ◽  
Media Composition ◽  
Microscopy Imaging ◽  
Biofilm Growth ◽  
Tetrazolium Chloride

Biofilms are multicellular communities of bacteria that can adhere to virtually any surface. Bacterial biofilms are clinically relevant, as they are responsible for up to two-thirds of hospital acquired infections and contribute to chronic infections. Troublingly, the bacteria within a biofilm are adaptively resistant to antibiotic treatment and it can take up to 1000 times more antibiotic to kill cells within a biofilm when compared to planktonic bacterial cells. Identifying and optimizing compounds that specifically target bacteria growing in biofilms is required to address this growing concern and the reported antibiofilm activity of natural and synthetic host defence peptides has garnered significant interest. However, a standardized assay to assess the activity of antibiofilm agents has not been established. In the present work, we describe two simple assays that can assess the inhibitory and eradication capacities of peptides towards biofilms that are formed by both Gram-positive and negative bacteria. These assays are suitable for high-throughput workflows in 96-well microplates and they use crystal violet staining to quantify adhered biofilm biomass as well as tetrazolium chloride dye to evaluate the metabolic activity of the biofilms. The effect of media composition on the readouts of these biofilm detection methods was assessed against two strains of Pseudomonas aeruginosa (PAO1 and PA14), as well as a methicillin resistant strain of Staphylococcus aureus. Our results demonstrate that media composition dramatically alters the staining patterns that were obtained with these dye-based methods, highlighting the importance of establishing appropriate biofilm growth conditions for each bacterial species to be evaluated. Confocal microscopy imaging of P. aeruginosa biofilms grown in flow cells revealed that this is likely due to altered biofilm architecture under specific growth conditions. The antibiofilm activity of several antibiotics and synthetic peptides were then evaluated under both inhibition and eradication conditions to illustrate the type of data that can be obtained using this experimental setup.

scholarly journals Direct Visualization of Amlodipine Intervention into Living Cells by Means of Fluorescence Microscopy

Molecules ◽  
2021 ◽  
Vol 26 (10) ◽  
pp. 2997
Author(s):  
Christine Quentin ◽  
Rūta Gerasimaitė ◽  
Alexandra Freidzon ◽  
Levon S. Atabekyan ◽  
Gražvydas Lukinavičius ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Aqueous Solutions ◽  
Time Lapse ◽  
Drug Uptake ◽  
Live Cells ◽  
Direct Visualization ◽  
Cellular Environment ◽  
Time Lapse Imaging ◽  
The Impact

Amlodipine, a unique long-lasting calcium channel antagonist and antihypertensive drug, has weak fluorescence in aqueous solutions. In the current paper, we show that direct visualization of amlodipine in live cells is possible due to the enhanced emission in cellular environment. We examined the impact of pH, polarity and viscosity of the environment as well as protein binding on the spectral properties of amlodipine in vitro, and used quantum chemical calculations for assessing the mechanism of fluorescence quenching in aqueous solutions. The confocal fluorescence microscopy shows that the drug readily penetrates the plasma membrane and accumulates in the intracellular vesicles. Visible emission and photostability of amlodipine allow confocal time-lapse imaging and the drug uptake monitoring.

scholarly journals Flower-like patterns in multi-species biofilms

2019 ◽  
Author(s):  
Liyang Xiong ◽  
Yuansheng Cao ◽  
Robert Cooper ◽  
Wouter-Jan Rappel ◽  
Jeff Hasty ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Phase Field Model ◽  
Time Lapse ◽  
Soft Agar ◽  
Local Concentration ◽  
Growth And Survival ◽  
Surface Time ◽  
Motile Species ◽  
Time Lapse Imaging ◽  
Motile Bacteria

AbstractDiverse interactions among species within bacterial biofilms often lead to intricate spatiotemporal dynamics. The spatial structure of biofilms can determine growth and survival of different species, but the mechanisms driving formation of this structure are not fully understood. Here, we describe the emergence of complex structures in a biofilm grown from mixtures of motile and non-motile bacterial species on a soft agar surface. Time-lapse imaging shows that non-motile bacteria “hitchhike” on the motile bacteria as the latter migrate outward. The non-motile bacteria accumulate at the boundary of the colony and trigger an instability that leaves behind striking flower-like patterns. The mechanism of the front instability governing this pattern formation is elucidated by a mathematical model for the frictional motion of the colony interface, with friction depending on the local concentration of the nonmotile species along the interface. A more elaborate two-dimensional phase-field model that explicitly accounts for the interplay between growth, mechanical stress from the motile species, and friction provided by the non-motile species, fully reproduces the observed flower-like patterns.

scholarly journals Single-Cell Analysis of the Dps Response to Oxidative Stress

2016 ◽  
Vol 198 (11) ◽  
pp. 1662-1674 ◽  
Author(s):  
Michela De Martino ◽  
Dmitry Ershov ◽  
Peter J. van den Berg ◽  
Sander J. Tans ◽  
Anne S. Meyer
Keyword(s):  
Oxidative Stress ◽  
Fluorescence Microscopy ◽  
Single Cells ◽  
Single Pulse ◽  
Time Lapse ◽  
Stress Conditions ◽  
Metal Exposure ◽  
Microscopy Imaging ◽  
Content Type ◽  
E Coli

ABSTRACTMicroorganisms have developed an elaborate spectrum of mechanisms to respond and adapt to environmental stress conditions. Among these is the expression ofdps, coding for theDNA-bindingprotein fromstarved cells. Dps becomes the dominant nucleoid-organizing protein in stationary-phaseEscherichia colicells and is required for robust survival under stress conditions, including carbon or nitrogen starvation, oxidative stress, metal exposure, and irradiation. To study the complex regulation of Dps inE. coli, we utilized time-lapse fluorescence microscopy imaging to examine the kinetics, input encoding, and variability of the Dps response in single cells. In the presence of an oxidative stressor, we observed a single pulse of activation of Dps production. Increased concentrations of H2O2led to increased intensity and duration of the pulse. While lower concentrations of H2O2robustly activated the Dps response with little effect on the growth rate, higher concentrations of H2O2resulted in dramatically lower and highly varied growth rates. A comparison of cells exposed to the same concentration of H2O2revealed that increased levels of Dps expression did not confer a growth advantage, indicating that recovery from stress may rely primarily upon variation in the amount of damage caused to individual cells.IMPORTANCEWe show for the first time the response of the DNA-binding protein from starved cells (Dps) to oxidative stress in single cells ofE. coli. Through time-lapse fluorescence microscopy, a single pulse of Dps production is observed in cells exposed to H2O2, with a duration and intensity of induction proportional to the concentration of the applied stress. More intense Dps expression did not provide a growth benefit to the bacteria, suggesting that healing from oxidative stress may largely depend upon the amount of damage in each individual cell.

scholarly journals Flower-like patterns in multi-species bacterial colonies

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Liyang Xiong ◽  
Yuansheng Cao ◽  
Robert Cooper ◽  
Wouter-Jan Rappel ◽  
Jeff Hasty ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Phase Field Model ◽  
Time Lapse ◽  
Soft Agar ◽  
Local Concentration ◽  
Growth And Survival ◽  
Surface Time ◽  
Motile Species ◽  
Time Lapse Imaging ◽  
Motile Bacteria

Diverse interactions among species within bacterial colonies lead to intricate spatiotemporal dynamics, which can affect their growth and survival. Here, we describe the emergence of complex structures in a colony grown from mixtures of motile and non-motile bacterial species on a soft agar surface. Time-lapse imaging shows that non-motile bacteria 'hitchhike' on the motile bacteria as the latter migrate outward. The non-motile bacteria accumulate at the boundary of the colony and trigger an instability that leaves behind striking flower-like patterns. The mechanism of the front instability governing this pattern formation is elucidated by a mathematical model for the frictional motion of the colony interface, with friction depending on the local concentration of the non-motile species. A more elaborate two-dimensional phase-field model that explicitly accounts for the interplay between growth, mechanical stress from the motile species, and friction provided by the non-motile species, fully reproduces the observed flower-like patterns.

Development of a PCR Assay for Detection of Enterobacteriaceae in Foods

2003 ◽  
Vol 66 (10) ◽  
pp. 1798-1804 ◽  
Author(s):  
SHIGERU NAKANO ◽  
TORU KOBAYASHI ◽  
KENICHI FUNABIKI ◽  
ATSUSHI MATSUMURA ◽  
YASUHIRO NAGAO ◽  
...  
Keyword(s):  
Salmonella Enteritidis ◽  
Enrichment Culture ◽  
Simple Procedure ◽  
Bacterial Species ◽  
Brain Heart Infusion ◽  
Rrna Gene ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Pcr Assay ◽  
Chelex 100

A broad-range PCR assay for the detection of bacteria belonging to the Enterobacteriaceae family was developed in this study. Primers targeting the bacterial 16S rRNA gene were newly designed and used in this PCR assay. To determine the specificity of the assay, 72 different bacterial species (of 49 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for every tested Vibrioaceae or Enterobacteriaceae strain except Proteus mirabilis. For all other bacterial strains and eukaryotes tested, results were negative. Bacterial DNA for PCR was prepared by a simple procedure with the use of Chelex 100 resin from culture after growth in brain heart infusion medium. To test this PCR assay for the monitoring of the Enterobacteriaceae family, either Escherichia coli or Salmonella Enteritidis was inoculated into various foods as an indicator. Prior to the PCR, the inoculation of 10 to 40 CFU of bacteria per g of food was followed by a 5-h enrichment culture step, and the PCR assay allowed the detection of bacterial cells. When actual examinations of the contamination of 15 noodle foods with Enterobacteriaceae by this PCR assay were conducted, 33% (5 of 15) of the samples tested positive. These results agreed with those of the Petri film Enterobacteriaceae Count Plate assay. Including the enrichment culture step, the entire PCR detection process can be completed within 7 h.

scholarly journals Development of a Multispecies Oral Bacterial Community in a Saliva-Conditioned Flow Cell

2004 ◽  
Vol 70 (7) ◽  
pp. 4340-4348 ◽  
Author(s):  
Jamie S. Foster ◽  
Paul E. Kolenbrander
Keyword(s):  
Spatial Organization ◽  
Bacterial Species ◽  
Fusobacterium Nucleatum ◽  
Flow Cell ◽  
In Vitro Models ◽  
Individual Species ◽  
Bacterial Strains ◽  
Symbiotic Relationships ◽  
Multispecies Biofilm

ABSTRACT Microbial communities within the human oral cavity are dynamic associations of more than 500 bacterial species that form biofilms on the soft and hard tissues of the mouth. Understanding the development and spatial organization of oral biofilms has been facilitated by the use of in vitro models. We used a saliva-conditioned flow cell, with saliva as the sole nutritional source, as a model to examine the development of multispecies biofilm communities from an inoculum containing the coaggregation partners Streptococcus gordonii, Actinomyces naeslundii, Veillonella atypica, and Fusobacterium nucleatum. Biofilms inoculated with individual species in a sequential order were compared with biofilms inoculated with coaggregates of the four species. Our results indicated that flow cells inoculated sequentially produced biofilms with larger biovolumes compared to those biofilms inoculated with coaggregates. Individual-species biovolumes within the four-species communities also differed between the two modes of inoculation. Fluorescence in situ hybridization with genus- and species-specific probes revealed that the majority of cells in both sequentially and coaggregate-inoculated biofilms were S. gordonii, regardless of the inoculation order. However, the representation of A. naeslundii and V. atypica was significantly higher in biofilms inoculated with coaggregates compared to sequentially inoculated biofilms. Thus, these results indicate that the development of multispecies biofilm communities is influenced by coaggregations preformed in planktonic phase. Coaggregating bacteria such as certain streptococci are especially adapted to primary colonization of saliva-conditioned surfaces independent of the mode of inoculation and order of addition in the multispecies inoculum. Preformed coaggregations favor other bacterial strains and may facilitate symbiotic relationships.

Cleavage of Human Embryos: Options and Diversity

Acta Naturae ◽  
2016 ◽  
Vol 8 (3) ◽  
pp. 88-96
Author(s):  
Yu. K. Doronin ◽  
I. V. Senechkin ◽  
L. V. Hilkevich ◽  
M. A. Kurcer
Keyword(s):  
Time Lapse ◽  
Reproductive Technologies ◽  
Human Embryos ◽  
Imaging Data ◽  
Noninvasive Methods ◽  
Topographic Features ◽  
Time Parameters ◽  
Embryo Cleavage ◽  
Time Lapse Imaging ◽  
Developmental Dynamics

In order to estimate the diversity of embryo cleavage relatives to embryo progress (blastocyst formation), time-lapse imaging data of preimplantation human embryo development were used. This retrospective study is focused on the topographic features and time parameters of the cleavages, with particular emphasis on the lengths of cleavage cycles and the genealogy of blastomeres in 2- to 8-cell human embryos. We have found that all 4-cell human embryos have four developmental variants that are based on the sequence of appearance and orientation of cleavage planes during embryo cleavage from 2 to 4 blastomeres. Each variant of cleavage shows a strong correlation with further developmental dynamics of the embryos (different cleavage cycle characteristics as well as lengths of blastomere cycles). An analysis of the sequence of human blastomere divisions allowed us to postulate that the effects of zygotic determinants are eliminated as a result of cleavage, and that, thereafter, blastomeres acquire the ability of own syntheses, regulation, polarization, formation of functional contacts, and, finally, of specific differentiation. This data on the early development of human embryos obtained using noninvasive methods complements and extend our understanding of the embryogenesis of eutherian mammals and may be applied in the practice of reproductive technologies.

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